Signal standardization of the secondary ion mass spectrometry (SIMS) microscope for quantification of halogens and calcium in biological applications

The secondary ion mass spectrometry (SIMS) microscope is able to map chemical elements in tissue sections. Although absolute quantification of an element remains difficult, a relative quantitative approach is possible for soft tissue by using carbon (¹²C) as an internal reference present at large homogeneous and constant concentration in specimen and embedding resin. In this study, this approach is used to standardize the signal of an SIMS microscope for the quantification of halogens (¹⁹F^—, ³⁵Cl^— and ⁷⁹Br^—) and calcium (⁴⁰Ca⁺). Standard preparation was determined based on homogeneity and stability criteria by molecular incorporation (halogens) or mixing (calcium) in methacrylate resin. Standard measurements were performed by depth analysis on areas of 8 μm (halogens) and 150 μm (calcium) in diameter for 10–30 min, under Cs⁺ (halogens) or O₂⁺ (calcium) bombardment. Results obtained from 100–120 measurements for each standard dilution show that the relationship between the signal intensity measured and the elemental concentration (μg/mg of wet tissue or mm ) is linear in the range of biological concentrations. This quantitative approach was applied firstly to bromine of the 5-bromo-2′-deoxyuridine (BrdU) used as nuclear marker of rat hepatocytes in proliferation. The second model concerns depletion of calcium concentration in cortical compartment in Paramecium tetraurelia during exocytosis. Then signal standardization in SIMS microscopy allows us to correlate quantitative results with those obtained from other methods.     

Morphological and elemental integrity of freeze-fractured,freeze-dried cultured cells during ion microscopic analysis

The effects of progressive ion beam bombardment on freeze-fractured, freeze-dried cultured cells during ion microscopic (SIMS) analysis were studied with scanning electron microscopy (SEM) and ion microscopy. The freeze-fracture, freeze-dry sample preparation method was generally found to preserve cell morphology to a level far exceeding the spatial resolution of the ion microscope, with splitting at the nuclear envelope being the most commonly observed artefact. SEM monitoring of surface topography of an NRK-49F fibroblast after various ion bombardment doses showed relatively uniform erosion of cellular material, with some apparent selective retention of small cytoplasmic granules. Prolonged bombardment produced no detectable lateral elemental translocation. ⁴¹K+/²⁴Mg+ signal ratios from Swiss 3T3 fibroblasts and RBL rat basophilic leukaemia cells were shown to vary generally by less than 10% during the course of extended ion bombardment. GM0415 human skin fibroblasts containing engorged lysosomes characteristic of Hurler's Syndrome were used to evaluate the effects of ion bombardment during a typical analysis session, where ion images of ³⁹K+, ²³Na+, ⁴⁰Ca+ and ²⁴Mg+ are sequentially recorded. This cell line was chosen as a worst-case system, because these cells are often thinly spread and possess extreme surface topography. Thin cell edges were shown sometimes to sputter away during analysis, giving misleadingly low ion signals from these regions in some ²⁴Mg+ micrographs. Various nonuniform sputtering phenomena occurring in the submicrometre spatial domain had little or no measurable impact on local intensities in ion micrographs, indicating that freeze-dried, freeze-fractured cells are sampled in a sufficiently uniform fashion that quantitative ion microscopic evaluations of intracellular elemental levels in the general cytoplasmic or nuclear regions are feasible.     

Evaluation of fracture planes and cell morphology in complementary fractures of cultured cells in the frozen-hydrated state by field-emission secondary electron microscopy: feasibility for ion localization and fluorescence imaging studies

We have employed field-emission secondary electron microscopy (FESEM) for morphological evaluation of freeze-fractured frozen-hydrated renal epithelial LLC-PK₁ cells prepared with our simple cryogenic sandwich-fracture method that does not require any high-vacuum freeze-fracture instrumentation (Chandra et al. (1986) J. Microsc. 144 , 15–37). The cells fractured on the substrate side of the sandwich were matched one-to-one with their corresponding complementary fractured faces on the other side of the sandwich. The FESEM analysis of the frozen-hydrated cells revealed three types of fracture: (i) apical membrane fracture that produces groups of cells together on the substrate fractured at the ectoplasmic face of the plasma membrane; (ii) basal membrane fracture that produces basal plasma membrane-halves on the substrate; and (iii) cross-fracture that passes randomly through the cells. The ectoplasmic face (E-face) and protoplasmic face (P-face) of the membrane were recognized based on the density of intramembranous particles. Feasibility of fractured cells was shown for intracellular ion localization with ion microscopy, and fluorescence imaging with laser scanning confocal microscopy. Ion microscopy imaging of freeze-dried cells fractured at the apical membrane revealed well-preserved intracellular ionic composition of even the most diffusible ions (total concentrations of K⁺, Na⁺ and Ca⁺). Structurally damaged cells revealed lower K⁺ and higher Na⁺ and Ca⁺ contents than in well-preserved cells. Frozen-freeze-dried cells also allowed imaging of fluorescently labelled mitochondria with a laser scanning confocal microscope. Since these cells are prepared without washing away the nutrient medium or using any chemical pretreatment to affect their native chemical and structural makeup, the characterization of fracture faces introduces ideal sample types for chemical and morphological studies with ion and electron microscopes and other techniques such as laser scanning confocal microscopy, atomic force microscopy and near-field scanning optical microscopy.     

̼���׹����˵��ȵ������ײ����е�Ӧ���о�

????о??????????????U⁺?????????????????CNTs??????????????????????????????????????????????????²³⁵U/²³⁸U????????????????о?????????????λ??????????????????????ī??????????????????????????????????????????????????????????????????U⁺?????????????????????????????????CNTs-3??Ч?????????????U⁺???????3?????????CNTs-3?????????????????25 ng???²³⁵U/²³⁸U????????С??0.2%??n=18?????????5 ng???²³⁵U/²³⁸U?????????0.5%??n=18????     

Automated focusing in bright-field microscopy for tuberculosis detection

Pulsed‐laser atom‐probe tomography is used to compare the field‐evaporation mass spectrum and spatial distribution of molecular fragments from various poly(3‐alkylthiophene) films deposited on sharpened aluminium specimen carriers using two different deposition methods. Films deposited via a modified solution‐cast methodology yield small fragments with a uniform structural morphology whereas films deposited via an electrospray ionization methodology yield a wide range of fragments with a very non‐uniform structural morphology. The main field‐evaporated chemical species identified for both deposition types were, in order of typical relative abundance, C₂H₅⁺, CH₃⁺, C₂H₄⁺, followed by C₃H_7,8⁺/SC⁺ and SCH⁺. Thick electrospray depositions allowed investigation of the influence of laser‐pulse energy on the analysis. Evidence is presented supporting the presence of a critical laser‐pulse energy whereby changes in film morphology are signalled by the appearance of a new mass fragment at 190 Da.     

Mass-separated microbeam system with a liquid-metal-ion source

This paper describes the developement of a mass-separated focusing column, consisting of a liquid-meta-ion (LMI) source, two lenses, an E×B mass filter, and a post-deflector. This system has the ability to produce sub-μm beams with energies up to 20 keV and a maximum current ranging from 10⁻² to 1 nA, depending on the source characteristics. Preliminary operation of this focusing system was demonstrated by scanning ion microscope (SIM) images. An image resolution of less than 0.1 μm has been achieved for focused beams of ⁶⁹Ga⁺ and ¹¹B⁺, which were respectively extracted from Ga-LMI and eutectic alloy LMI sources.     

Evaluation of a liquid metal ion source for secondary ion mass spectrometry

Positive and negative secondary ion emission of 23 pure elements have been studied for 10 keV In⁺ and 10 keV O₂⁺ bombardment. In⁺ ions were produced in a liquid metal ion source. For most of the elements investigated positive and negative secondary ion yields under In⁺ impact are comparable to those obtained with O₂⁺ primary ions. Admission of oxygen into the sample chamber enhances positive and negative ion intensitities in a strongly element-specific manner. Depth profiles of a Ni/Cr multilayer (100 Å single-layer thickness) using 5 keV In⁺ primary ions show that these ions may also be applied successfully for secondary ion mass spectrometric depth profiling.     

Quantitative subcellular imaging of boron compounds in individual mitotic and interphase human glioblastoma cells with imaging secondary ion mass spectrometry (SIMS)

Boron measurements at subcellular scale are essential in boron neutron capture therapy (BNCT) of cancer as the nuclear localization of boron‐10 atoms can enhance the effectiveness of killing individual tumour cells. Since tumours contain a heterogeneous population of cells in interphase as well as in the M phase (mitotic division) of the cell cycle, it is important to evaluate the subcellular distribution of boron in both phases. In this work, the secondary ion mass spectrometry (SIMS) based imaging technique of ion microscopy was used to quantitatively image boron from two BNCT agents, clinically used p‐boronophenylalanine (BPA) and 3‐[4‐(o‐carboran‐1‐yl)butyl]thymidine (N4), in mitotic metaphase and interphase human glioblastoma T98G cells. N4 belongs to a class of experimental BNCT agents, designated 3‐carboranyl thymidine analogues (3CTAs), which presumably accumulate selectively in cancer cells due to a process referred to as kinase‐mediated trapping (KMT). The cells were exposed to BPA for 1 h and N4 for 2 h. A CAMECA IMS‐3f SIMS ion microscope instrument capable of producing isotopic images with 500 nm spatial resolution was used in the study. Observations were made in cryogenically prepared fast frozen, and freeze‐fractured, freeze‐dried cells. Three discernible subcellular regions were studied: the nucleus, a characteristic mitochondria‐rich perinuclear cytoplasmic region, and the remaining cytoplasm in interphase T98G cells. In metaphase cells, the chromosomes and the cytoplasm were studied for boron localization. Intracellular concentrations of potassium and sodium also were measured in each cell in which the subcellular boron concentrations were imaged. Since the healthy cells maintain a K/Na ratio of approximately 10 due to the presence of Na‐K‐ATPase in the plasma membrane of mammalian cells, these measurements provided validation for cryogenic sample preparation and indicated the analysis healthy, well preserved cells. The BPA‐treated interphase cells revealed significantly lower concentrations of boron in the perinuclear mitochondria‐rich cytoplasmic region as compared to the remaining cytoplasm and the nucleus, which were not significantly different from each other. In contrast, the BPA‐treated metaphase cells revealed significantly lower concentration of boron in their chromosomes than cytoplasm. In addition, the cytoplasm of metaphase cells contained significantly less boron than the cytoplasm of interphase cells. These observations provide valuable information on the reduced uptake of boron from BPA in mitotic cells for BPA‐mediated BNCT. SIMS observations on N4 revealed that boron was distributed throughout the interphase and mitotic cells, including the chromosomes. The presence of boron in chromosomes of metaphase cells treated with N4 is indicative of a possible incorporation of this thymidine analogue into DNA. The 3‐D SIMS imaging approach for the analysis of mitotic cells shown in this work should be equally feasible to the evaluation of other BNCT agents.     

An X-ray microanalytical examination of precipitation methods for the ultrastructural localization of potassium in plant tissue: I. Cobaltinitrite

In vitro and in vivo studies were carried out to assess the feasibility of adapting the cobaltinitrite method for the ultrastructural localization of K⁺ in plant cells. The relatively low mobility of the cobaltinitrite ion and the solubility of the precipitation product resulted in the formation of only a few large deposits in each cell. The addition of Ag⁺ at the time of precipitation was found to increase the number of small deposits retained in sections. However, cobaltinitrite reagents frequently caused plasmolysis and poor cytoplasmic preservation. Precipitation during freeze-substitution was also attempted. It was concluded that precipitation of K⁺ using cobaltinitrite has only limited use in low magnification electron microscopy in conjunction with light microscopy.     

Methods of quantitative matrix analysis of Zircaloy-2

The zirconium-based alloy Zircaloy-2 contains small amounts of iron, chromium and nickel dissolved in the matrix. Several attempts to measure these amounts have been made in the past, but the results are conflicting and inconclusive. The advent of wide angle, laser pulsed atom probe tomography motivates a new attempt to analyze the matrix. Large datasets are now easily obtained using laser pulsing but quantification is not straightforward due to rather complex mass spectra. Zircaloy-2 contains about 1 wt% tin, 0.1 wt% oxygen and trace amounts of Si, C and Al. Severe overlaps make quantification of any Fe⁺, Cr⁺ and Ni⁺ ions impossible. Quantification of Fe, Cr and Ni therefore requires that they appear as doubly charged ions only, and consequently the field must be kept high enough. In addition, adsorbed CO⁺ may appear at the main peak of Fe²⁺. In the paper a method is reported, which gives what we believe an accurate quantitative analysis of at least iron and chromium in the matrix.     

The use of the stable isotope 44Ca in studies of calcium incorporation into dentin

The incorporation into rat incisor dentin of two calcium isotopes, the stable ⁴⁴Ca and the radioactive ⁴⁵Ca, was studied using secondary ion mass spectrometry (SIMS) stepscanning and imaging, and autoradiography, respectively. The results demonstrated a time-dependent incorporation of the calcium isotopes into the mineral phase of dentin. With the SIMS step-scanning, detecting ⁴⁴Ca, the ion yield was high in the odontoblasts 2 min after intravenous injection. After 10 min a marked increase in signal intensity was found at the dentin mineralization front. This result was consistent with those obtained by ⁴⁵Ca autoradiography; a peak of incorporation occurred 10 min after injection of the isotope. Likewise, localization of ⁴⁴Ca to the mineralization front could be demonstrated 10 min after injection by SIMS imaging. In images obtained at earlier intervals, no such increase in ion yield could be detected. The results show that the nonradioactive, stable isotope ⁴⁴Ca can be used as a marker for biomineralization in a similar way to radioactive ⁴⁵Ca.     

Quantitative secondary ion mass spectrometry imaging of self-assembled monolayer films for electron beam dose mapping in the environmental scanning electron microscope

Fluorinated alkanethiol self-assembled monolayers (SAM) films immobilized on gold substrates have been used as electron-sensitive resists to map quantitatively the spatial distribution of the primary electronbeam scattering in an environmental scanning electron microscope (ESEM). In this procedure, a series of electron dose standards are prepared by exposing a SAM film to electron bombardment in well-defined regions at different levels of electron dose. Microbeam secondary ion mass spectrometry (SIMS) using Cs⁺ bombardment is then used to image the F^- secondary ion signal from these areas. From the reduction in F^- intensity as a function of increasing electron dose, a calibration curve is generated that allows conversion of secondary ion signal to electron dose on a pixel-by-pixel basis. Using this calibration, electron dose images can be prepared that quantitatively map the electron scattering distribution in the ESEM with micrometer spatial resolution. The SIMS imaging technique may also be used to explore other aspects of electron-surface interactions in the ESEM.     

Ion microscopy imaging of various pyrimidic nucleotides and analogs in the nuclei of cultured tumor cells

New applications for ion microscopy are presented. This method has been used primarily to detect mineral elements (K⁺, Na⁺, AL⁺). It also can be used to detect organic molecules containing halogen atoms and radioactive isotopes. ¹⁴C-nucleotides and halogenated pyrimidic nucleotides and analogs were employed in this study. The various images obtained were correlated with the mechanism of action of these compounds, thus opening new lines of research.     

Retaining ionic concentrations during in vitro storage of tissue for microanalytical studies

When human or animal tissue is to be investigated by X-ray microanalysis, it is sometimes necessary to store the tissue between removal from the organism and freezing. However, when excised tissue is stored in buffer, the elemental concentrations in the cell may change. In the present study, it was attempted to develop a storage buffer that would retain the cellular elemental concentrations close to their in situ values. To start, the NaCl component in Krebs–Ringer buffer was exchanged for K-gluconate and KCl for NaCl. This buffer was called a ‘100% high K⁺ solution’. Starting from this solution, part of the K-gluconate was replaced by an equivalent amount of NaCl. Incubation of excised rat liver (4 °C, 4 h) in 85% high K⁺ solution resulted in retention of cellular Na, K, Ca, S and Mg concentration most closely to the in situ state, whereas cellular Cl was retained best when the tissue was incubated in 75% high K⁺ solution. For rat submandibular gland, incubation in 80% high K⁺ solution resulted in optimal retention of cellular Na, K, Ca, P, S and Mg, while Cl was retained best in a 70% high K⁺ solution. Based on these results, an optimally modified Krebs–Ringer solution for the liver would consist of 119 mM K⁺, 26 mM Na⁺ and 45 mM Cl^?. An optimally modified Krebs–Ringer bicarbonate solution for the submandibular gland would be composed of 96 mM K⁺, 53 mM Na⁺ and 46 mM Cl^?. After incubation in the modified solutions (at 4 °C), cellular Na, Mg, S, Cl, K and Ca in both tissues were maintained close to the in situ state throughout a 6-h incubation. The cellular P concentration was reduced after incubation for 1 h; thereafter, in the liver cells it remained at this lower level for the rest of the incubation, whereas in the submandibular gland tissue it increased again after 4 h. The increase in cell volume (oedema) was less in tissue stored in the modified solutions, than in the 100% high K⁺ solutions. Incubation in high Na⁺ buffers (4 °C, 6 h) resulted in a progressive increase in the percentage of cells showing trypan blue uptake. A similar increase in trypan blue uptake was seen in the modified solution, but this increase levelled off after 4 h. After cholinergic stimulation in high Na⁺ solution (25 °C, 1 min), the expected decrease in cellular Cl concentration was seen in submandibular gland cells that had previously been preserved (4 °C, 4 h) in the modified solution, but not in those that had been preserved in the 100% high K⁺ solution.     

Imaging intracellular elemental distribution and ion fluxes in cultured cells using ion microscopy: a freeze-fracture methodology

A freeze-fracture methodology was standardized for tissue culture cells to study intracellular distribution of diffusible elements with ion microscopy. Chinese hamster ovary (CHO) and normal rat kidney (NRK) cells grown on a silicon substrate were sandwiched using another smooth surface (silicon, glass, mica) in the presence of spacers and fast frozen in liquid nitrogen slush. The sandwich was fractured by prying the two halves apart under liquid nitrogen. This procedure produced large areas on the silicon substrate containing hundreds of cells grouped together and fractured at the apical cell surface. After freeze-drying, these cells revealed a subcellular distribution of Na, K, Ca, Mg, P, Cl and S with the ～0·5 μm lateral resolution of the ion microscope. Between the nuclei and the cytoplasm of cells, no major differences were observed for Na, K, Mg, P, Cl and S intensities. Calcium alone, however, exhibited a remarkable distribution. Calcium accumulated more in the cytoplasm than in the nuclei of cells. Even within the cytoplasm its distribution was heterogeneous, suggesting Ca binding sites. The fractured cells consistently exhibited high K-low Na intensities. The injured or dead cells were easily recognized among the healthy ones due to their abnormal ion composition. This simple freeze-fracture methodology allowed fracturing of cells without removing the cells from the substrate. In addition, it eliminated the need for washing the nutrient media away and cryo-sectioning before ion microanalysis. The methodology was successfully extended to 3T3 mouse fibroblast, PtK₂ rat kangaroo and L5 rat myoblast cultures.     

Influence of ion bombardment on tribological properties of UHMWPE

Medical grade UHMWPE-s: GUR 1020 and 1050, were subjected to ion bombardment with H₂⁺, He⁺, Ar⁺ or Ag⁺ of different energy and various doses. The work presents changes to micromechanical profiles and surface morphology (AFM, SEM) of polyethylenes due to the treatment. Mechanisms behind the modification have been proposed owing to structural and chemical analyses by s-SIMS, FTIR-IRS, confocal Raman microscopy and “nuclear depth profiling”. Studies revealed that hardness of the surface layer increases due to radiolysis, probably leading to cross-polymerization of polyethylene. The depth of modification is limited to the penetration of ion beam, being dependent on a dose and a kind of ions applied. The treatment results additionally in oxidation, which together with a development of the surface geometry is responsible for its hydrofilization and, in some cases bacteriostatic character. Friction and wear of polyethylenes under a high load, adequate to extreme conditions of exploitation of hip joints, can be reduced even by three times, due to a proper ion beam treatment.     

Limitations of beam damage in electron spectroscopic tomography of embedded cells

Elemental mapping in the energy filtering transmission electron microscope (EFTEM) can be extended into three dimensions (3D) by acquiring a series of two‐dimensional (2D) core‐edge images from a specimen oriented over a range of tilt angles, and then reconstructing the volume using tomographic methods. EFTEM has been applied to imaging the distribution of biological molecules in 2D, e.g. nucleic acid and protein, in sections of plastic‐embedded cells, but no systematic study has been undertaken to assess the extent to which beam damage limits the available information in 3D. To address this question, 2D elemental maps of phosphorus and nitrogen were acquired from unstained sections of plastic‐embedded isolated mouse thymocytes. The variation in elemental composition, residual specimen mass and changes in the specimen morphology were measured as a function of electron dose. Whereas 40% of the total specimen mass was lost at doses above 10⁶ e^?/nm², no significant loss of phosphorus or nitrogen was observed for doses as high as 10⁸ e^?/nm². The oxygen content decreased from 25 ± 2 to 9 ± 2 atomic percent at an electron dose of 10⁴ e^?/nm², which accounted for a major component of the total mass loss. The specimen thickness decreased by 50% after a dose of 10⁸ e^?/nm², and a lateral shrinkage of 9.5 ± 2.0% occurred from 2 × 10⁴ to 10⁸ e^?/nm². At doses above 10⁷ e^?/nm², damage could be observed in the bright field as well in the core edge images, which is attributed to further loss of oxygen and carbon atoms. Despite these artefacts, electron tomograms obtained from high‐pressure frozen and freeze‐substituted sections of C. elegans showed that it is feasible to obtain useful 3D phosphorus and nitrogen maps, and thus to reveal quantitative information about the subcellular distributions of nucleic acids and proteins.     

Enhancing image contrast of carbon nanotubes on cellular background using helium ion microscope by varying helium ion fluence

Carbon nanotubes (CNTs) have become an important nano entity for biomedical applications. Conventional methods of their imaging, often cannot be applied in biological samples due to an inadequate spatial resolution or poor contrast between the CNTs and the biological sample. Here we report a unique and effective detection method, which uses differences in conductivities of carbon nanotubes and HeLa cells. The technique involves the use of a helium ion microscope to image the sample with the surface charging artefacts created by the He⁺ and neutralised by electron flood gun. This enables us to obtain a few nanometre resolution images of CNTs in HeLa Cells with high contrast, which was achieved by tailoring the He⁺ fluence. Charging artefacts can be efficiently removed for conductive CNTs by a low amount of electrons, the fluence of which is not adequate to discharge the cell surface, resulting in high image contrast. Thus, this technique enables rapid detection of any conducting nano structures on insulating cellular background even in large fields of view and fine spatial resolution. The technique demonstrated has wider applications for researchers seeking enhanced contrast and high‐resolution imaging of any conducting entity in a biological matrix – a commonly encountered issue of importance in drug delivery, tissue engineering and toxicological studies.     

碰撞池-多接收电感耦合等离子体质谱分析硅同位素的方法研究

通过更换仪器进样系统、改变溶液基体、调节碰撞气的种类和流量以及调节质量分辨率等参数,建立了碰撞池-多接收电感耦合等离子体质谱（Collision Cell-MC-ICP-MS）测定硅同位素丰度组成的分析方法。实验结果表明:使用PFA雾化器、Pt锥和蓝宝石材质的矩管可有效降低仪器本底;调节狭缝宽度、中心杯位置等可提高仪器的质量分辨率,实现硅同位素离子与同量异位素干扰离子在一定程度上的分离;选择氖气（Ne）和氩气（Ar）的混合气作为碰撞气、降低溶液中四甲基氢氧化铵（TMAH）浓度等能有效降低多原子离子的干扰。在此基础上,分析了样品WASO 04和GBW(E)080272中硅同位素的丰度组成,同位素丰度比R_30/28的测量精度达到0.02%。将样品WASO 04作为参考标准时,样品GBW(E)080272中的硅同位素分馏系数δ³⁰Si为-1.57‰。     

Effects of laser pulsing on analysis of steels by atom probe tomography

When performing atom probe tomography analysis, laser pulsing was found very helpful for some types of steels, which are prone to premature sample failure under voltage pulsing. Rather accurate chemical compositions for both matrix and precipitates were obtained by voltage- and laser-pulsed mode. Some special issues on the effects of laser are discussed. A simple correction based on the ¹³Cⁿ⁺ and ¹⁰Bⁿ⁺ peak was used to quantify C in the carbide (M₂₃C₆, M=Fe, Cr, Mo) and B in the boride (M₃B₂, M=Mo, Fe, Cr, V). The mass resolution in laser mode is sufficient to resolve ⁵⁶Fe²⁺ and ¹⁴N₂¹⁺ at 28 Da. Small peak shifts were found in the spectrum due to reflectron aberrations.